The mechanism of interaction of thiamin pyrophosphate, M2 ion and substrates with transketolase of baker's yeast is being investigated. Essential binding sites are identified by selective destruction of amino acid residues with phenylglyoxal, trinitrobenzene sulfonate, formaldehyde-NaBH3CN and photooxidation in the presence of rose bengal and methylene blue. In addition the ability of substrates, coenzymes and analogues to protect against these agents will give information about the specific binding sites. Inactivation with radioactive phenylglyoxal and amino acid analysis before and after various methods of inactivation will permit quantitative conclusions to be drawn. The nature of the binding, and the similarity or differences between the two binding sites for the coenzymes will be investigated by spin-echo NMR studies: proton relaxation rates will be measured at two or more frequencies with Mn2+ as the cation. Spin-labeled analogues of the substrates are being prepared. These will be used in EPR studies in which the distance between the substrate and the metal-coenzyme will be determined from measurement of the interactions of the two electron spins.